Collagenic material useful in particular for preventing post-operative adhesions

ABSTRACT

The present invention relates to a biocompatible collagenous material which is non-toxic and biodegradable in less than one month, preferably in less than one week. This material comprises collagen and at least one hydrophilic macromolecular additive which is chemically non-reactive towards the collagen, with the collagen having at least partially lost its helical structure and being crosslinked.  
     The invention also relates to a process for obtaining such a material.  
     The collagenous material according to the invention is used, in particular, for preventing post-operative adhesions.

[0001] The present invention relates to a biocompatible, non-toxiccollagenous material which is potentially adherent and rapidlybiodegradable. It relates more specifically to a collagenous materialwhich is able to inhibit the formation of post-operative adhesions.

[0002] The invention also relates to a process for obtaining such amaterial.

[0003] Post-operative adhesions develop following surgical interventionin a patient, certain of whose organs have been subjected to traumaengendered by the surgical act itself.

[0004] The wounds created by dissecting organs or tissues, or by anyother intervention, are characterized by two phenomena:

[0005] these wounds are not watertight and spontaneously exude a plasmafluid containing fibrin, sometimes even blood, if perfect haemostasishas not been achieved; certain organs ooze other liquids such as liverbile or cerebrospinal fluid in the case of the nervous system.

[0006] the damaged surfaces no longer possess an organized and stabletissue barrier and are occasionally even strewn with foreign bodies:suture threads, staples, blood clots, infectious agents, or tissuesburnt by diathermy knives or laser beams.

[0007] These two characteristics bring about a reaction in the organismwhich starts with inflammation and continues with an intense andgenerally poorly organized migration and proliferation of cells. Theseusually give rise to neo-formed, fibrous, vascularized tissues whichconnect the damaged organs to adjacent organs.

[0008] Many reviews have been published on the subject (see, inparticular, DI ZEREGA (1994)).

[0009] A variety of solutions have already been proposed, particularlyat the pharmacological level, with varying degrees of success.

[0010] The most effective solution known to date is to place on thewound a physical barrier which isolates the organs from each other andwhich allows them to cicatrize independently without developinginterrelationships.

[0011] Artificial, non-degradable tissues, to which cells do not attach,constitute the physical barriers which lead to the best results. Teflon®and silicone are examples of the most efficient polymers.

[0012] The major drawback is that these physical barriers have to beremoved, several weeks later, by means of a second surgical operationwhich can, furthermore, itself give rise to other secondary adhesions atthe site of the laparotomy.

[0013] It is therefore vital to develop biodegradable barriers whichavoid the necessity of a second intervention.

[0014] With this aim in mind, a large number of natural biodegradablepolymers made from gelatin, collagen, polysaccharides,mucopolysaccharides, etc. have already been proposed; however, thesepolymers have not led to satisfactory results.

[0015] According to RODEHEAVER's team (HARRIS et al., 1995), the minimumobjective is that the barrier should remain in place for at least 36hours. This is explained by the fact that a minimum time is required toenable the complex mechanisms of cicatrization to take their course.

[0016] However, the resorption time should not be too long. If thematerial remains in contact with the wound for several weeks it can giverise to persistent inflammatory reactions which favour a disorganizedand more substantial fibrous reaction and which can cause anatomicalproblems locally: thickness, rigidity, shrinkage, ischaemia, granulomaor a persistent focus of infection, particularly in contact with theintestines and the digestive organs.

[0017] Furthermore, it appears that the material which is to serve as aphysical barrier should adhere correctly to the damaged tissues, inparticular when protecting organs which are mobile or which aresubjected to variable distension, such as the intestine, or simply toprevent the barrier migrating in response to the movements of thepatient and to the mechanical constraints which these movements entail.

[0018] The difficulty of obtaining all these properties combined in oneand the same biomaterial explains why the solutions and productsproposed to date are still found to be very inadequate.

[0019] The biodegradable products which are currently marketed forpreventing adhesions are only partially active and yield results whichare still inadequate.

[0020] The cellulose derivatives which are marketed by Johnson & JohnsonMedical (Arlington, Tex., United States), such as the productsSURGICELL® or INTERCEED®, cannot be used in the presence of blood andgive disappointing results in some animal models (HARRIS et al., 1995).

[0021] The product SEPRAFILM®, which is marketed by Genzyme, (Cambridge,Mass., United States), is a film composed of hyaluronic acid andcarboxymethyl cellulose and which only appears to be effective in 50% ofcases (BECKER et al., 1996). While it degrades very rapidly in a fewdays, it is difficult to manipulate. It is fragile and brittle andimpossible to use through a trocar in association with laparoscopy.Finally, it progressively loses its initial adherence and can migratesome distance, thereby leaving the wound unprotected.

[0022] The products which have been recently described by researchworkers in the literature or in published patent applications stillappear to leave a large number of problems unresolved.

[0023] Products which have been recently proposed include a variety ofcollagenous materials.

[0024] KHOURY et al. (1994) propose, on behalf of COLETICA (Lyon,France), a collagen membrane which consists of two layers, with onelayer being based on native collagen and forming the support and beingcovered with a second layer of gelatin. The idea for this membrane isinspired by a visceral surgery patch which was developed by TAYOT et al.(1988). This material has a degradation time which is very much longerthan one month and does not, therefore, possess the essential property,which is that of disappearing very rapidly. It also suffers from thedrawback of not being sufficiently adherent and, as a consequence, ofhaving to be applied with suture stitches, which can give rise toundesirable complications.

[0025] In 1994, ORLY I. proposed, on behalf of COLETICA, a transparentcollagen membrane which consists of a single layer or of two layers,depending on the examples, and is usually combined with an undegradableprosthesis for treating hernias or eventrations. This membrane consistsof undenatured native collagen. It has to be stapled or sutured. Inorder to make the stapling easier, it is necessary for the membrane tobe transparent in order to avoid injuring sensitive zones (nerves andblood vessels), as in the case of inguinal hernias. The risks associatedwith stapling hernial prostheses, in particular, are well known. Forthis reason, surgeons will be more interested in gluing theseprostheses, thereby avoiding any sutures or staples. ORLY I. specifies,surprisingly, that this membrane should not be adhesive since it is notpossible to use the membrane developed by KHOURY et al., in the sameCompany, i.e. COLETICA (cited above), in this indication due to itsadhesive properties, which hinder insertion in association withabdominal surgery. The time taken for resorption in the case of thismembrane is considerably longer than one month since resorption is notcomplete at 5 weeks and 3 months are required for the material to havebeen almost totally eliminated. Despite these very long periods, theApplicant asserts, paradoxically, that the material has the advantage ofa “relatively” rapid resorption time.

[0026] In 1995, YEUNG et al. proposed, on behalf of Collagen Corporation(Palo Alto, Calif., United States), linking an anti-adhesion agentderived from polyethylene glycol to a collagenous substrate by means ofcovalent bonds. This product is very complicated to produce and requiresthe use of polyethylene glycol derivatives which are difficult tomanufacture, expensive and not devoid of toxic risks associated withtheir chemical reactivity. Finally, the resorption time which isindicated for this material consisting of dense collagen is usually from30 to 50 days.

[0027] In 1994, TARDY et al. proposed, in a patent on behalf of IMEDEX,now called SADUC, a liquid biological glue which is derived fromcollagen and which has anti-adhesion properties. The particularadvantages of this product are its adherence and its rapid degradationwithin a few days. The adhesive properties stem from the crosslinking ofthe molecules of collagen, which is oxidized with periodic acid andwhich is stored liquid at acid pH. This crosslinking is triggered byadding an alkaline solution, or a buffer, which neutralizes the pH andwhich rapidly transforms the solution of oxidized collagen into anadherent solid.

[0028] Nevertheless, the efficacy of this product in preventingpost-operative adhesions, which has been demonstrated in several animalmodels, is not easy to exploit in practice. Thus, this product has to bestored permanently in frozen form, something which is difficult toachieve in current hospital systems.

[0029] Various collagenous biomaterials obtained by crosslinkingcollagen following oxidation with periodic acid were also described byM. TARDY et al. in a patent in 1986. Films of crosslinked oxidizedcollagen were prepared by drying solutions of oxidized collagen, towhich glycerol had been added, under a stream of sterile air. Thesefilms have properties which depend on the quantity of glycerol employed.In order to obtain pliable, non-brittle films which possess acceptableresistance, a sufficient quantity of glycerol, approximately equal inweight to the quantity of collagen, has to be used. However, under theseconditions, the films stick to their support and to the surgeon's glovesand instruments, lack rigidity because of their delicacy and are verydifficult to use if they are not combined with another material.

[0030] The invention described below makes it possible to resolve thepreviously mentioned drawbacks.

[0031] The particular object of the invention is to provide abiocompatible material which is non-toxic and non-sticky to the touch inthe dry state for ease of manipulation, but which is able to developadhesive properties in an aqueous medium, in particular in aphysiological medium.

[0032] Another object of the invention is to provide a material which israpidly biodegradable, namely in less than one month and, preferably, inless than one week.

[0033] Another object of the invention is to provide a collagenousmaterial which is suitable for use as a post-operative anti-adhesionbarrier.

[0034] Another object of the invention is to provide a process forobtaining such a material.

[0035] To this end, the invention relates to a biocompatible collagenousmaterial which is non-toxic and biodegradable in less than one month,characterized in that it comprises collagen, which has at leastpartially lost its helical structure, and at least one hydrophilicmacromolecular additive which is chemically non-reactive towards thecollagen, with the collagen being crosslinked.

[0036] The invention also relates to such a material which isbiodegradable within less than one week.

[0037] The invention also relates to a process for obtaining abiocompatible collagenous material which is non-toxic, potentiallyadherent and biodegradable in less than one month, preferably in lessthan one week, characterized in that it comprises:

[0038] a) preparing a collagen solution;

[0039] b) treating the said solution in order to cause the collagen atleast partially to lose its helical structure;

[0040] c) mixing the resulting collagenous solution with a solutioncontaining at least one hydrophilic macromolecular additive which ischemically non-reactive towards the collagen which is present;

[0041] d) crosslinking the said mixture in order to obtain the desiredcollagenous material.

[0042] The inventors discovered, surprisingly, that a mixture ofcollagen, which had been denatured by moderate heating, and ahydrophilic macromolecular additive was able, after the collagen hadbeen crosslinked, to form a biocompatible and non-toxic material whichwas, at one and the same time, non-sticky to the touch in the dry state,adherent in an aqueous (physiological) medium, and biodegradable in afew days or a few weeks.

[0043] They demonstrated that it was possible to obtain such a materialby crosslinking the collagen, which had been previously modified byoxidative cleavage and heating, in the presence of a hydrophilicmacromolecular additive which was chemically non-reactive towards thecollagen.

[0044] While the function of the oxidative cleavage of the collagen isto permit subsequent moderate crosslinking of the collagenous material,the invention does not exclude the possibility of achieving thisfunction of moderate crosslinking by other means, for example by beta orgamma radiation, or using other agents for achieving moderatecrosslinking, for example using chemical agents in amounts which aresufficiently low and non-toxic.

[0045] While the treatment consisting of heating the collagen solutionat a temperature greater than 37° C. leads to the progressive loss ofthe helical structure of the collagen, the invention does not excludethe possibility that this function can be achieved by other physical orchemical means, for example by means of ultrasonication, or by means ofadding chaotropic agents.

[0046] The inventors discovered, in particular, that the presence of thehydrophilic additive unexpectedly made it possible to increase thedensity and mechanical resistance of the collagen and to render thecollagen potentially adhesive, that is to say to permit satisfactoryadhesiveness, in particular in a physiological medium, to organs and topromote degradation of the collagen, which degradation can thus beaccomplished, according to the embodiments of the invention, in lessthan seven days or less than 4 weeks.

[0047] The process for preparing a collagenous material according to thepresent invention is described below.

[0048] The collagen which is used for obtaining a collagenous materialas previously defined can equally well be of animal or human origin orobtained by means of genetic recombination. Use is preferably made ofnative collagen which has been solubilized at acid pH or after digestivetreatment with pepsin. The collagen can, in particular, be bovine type Icollagen or human type I or type III collagen or else mixtures of thelatter collagens in any proportions.

[0049] According to one embodiment of the invention, the collagen ismodified by means of oxidative cleavage. Periodic acid or one of itssalts can be used for this purpose, in accordance with the techniquedescribed by M. TARDY et al. (1986).

[0050] It may be recalled, briefly, that this technique consists insubjecting an acid solution of collagen to the action of periodic acidor one of its salts by mixing the collagen solution with a solution ofthis acid or salt at a concentration of between 1 and 10⁻⁵ M, preferablybetween 5×10⁻³M and 10⁻¹M, at a temperature in the vicinity of ambienttemperature and for a period which can range from 10 minutes to 72hours.

[0051] According to the invention, an acid solution of collagen is usedwhose concentration is between 5 and 50 g/l. The concentration ofcollagen is preferably 30 g/l.

[0052] This treatment induces cleavages in certain constituents of thecollagen, i.e. hydroxyproline and sugars, and thus creates reactivesites without thereby inducing crosslinking.

[0053] The oxidized collagen, which has thus been prepared in solution,is heated at a temperature greater than 37° C., preferably at atemperature of between 40 and 50° C. This results in the helicalstructure of the collagen being at least partially denatured.

[0054] The collagen is then crosslinked in the presence of at least onehydrophilic macromolecular additive which is chemically non-reactivetowards the collagen.

[0055] “Chemically non-reactive towards the collagen” is understood asmeaning a hydrophilic compound which is not able to react with thecollagen which is present and which in particular does not form anycovalent bond with the collagen while it is being crosslinked.

[0056] The hydrophilic macromolecular additive according to theinvention advantageously has a molecular weight which is greater than3000 daltons.

[0057] The hydrophilic additive can be a synthetic hydrophilic polymeradvantageously having a molecular weight of between 3000 and 20,000daltons. Polyethylene glycol is particularly preferred.

[0058] The hydrophilic additive can also be a polysaccharide, amongwhich may be mentioned starch, dextran and cellulose, which arepreferred.

[0059] It is also possible to envisage using those polysaccharides,which, in oxidized form, display carboxylic functions in thesemolecules.

[0060] While mucopolysaccharides may also be suitable for the aims ofthe invention, they are not preferred because their distinctive animalorigin renders them difficult to prepare while satisfying the prescribedstandards of traceability.

[0061] The hydrophilic additive is selected according to a variety ofparameters which are linked, in particular, to its use, such as itsprice, its harmlessness, its biodegradability and/or its ability to beeliminated, in particular via the kidneys, in the event of therapeuticuse.

[0062] The said crosslinking is achieved by mixing, at neutral pH, asolution of collagen, which has been modified by oxidative cleavage andheating as indicated above, with a solution containing at least onehydrophilic macromolecular additive.

[0063] The concentration of hydrophilic additive(s) is from 2 to 10times lower than that of the collagen.

[0064] The crosslinking of the collagen in the presence of thehydrophilic additive is effected at a temperature of between 4 and 30°C., preferably at from 18 to 25° C.

[0065] According to one embodiment, the crosslinking is envisaged to becarried out in the presence of glycerol, which can be added to thecollagen/hydrophilic additive mixture. In this case, the concentrationof glycerol is advantageously between 3 and 8 g/l, and does not exceedone third of the concentration of the collagen.

[0066] The polymerization takes place while the material is drying.

[0067] According to the invention, it is thought, without it beingpossible for this to be regarded as being limiting, that the hydrophilicadditive which is present during the crosslinking of the collagen iscontained in the collagenous network which is formed, even though itdoes not itself react with the collagen.

[0068] According to the envisaged applications, the crosslinkedcollagenous material can be subjected to a variety of standardtreatments such as drying, sterilization, etc.

[0069] The crosslinked collagen can be dried, in particular, in a streamof sterile air, if this is required.

[0070] Sterilization is advantageously implemented by means ofirradiation with beta radiation (irradiation with electrons) or gammaradiation (irradiation using radioactive cobalt).

[0071] In accordance with the invention, the collagenous material can beprepared in the form of a film, a gel or a paste.

[0072] According to the applications which are envisaged, thecollagenous material is advantageously conceived in the form of a film.

[0073] According to one particularly preferred embodiment, the solutioncontaining the collagen, which has at least partially lost its helicalstructure, in particular as a result of heating, and, where appropriate,has been modified by oxidative cleavage, a hydrophilic macromolecularadditive and, where appropriate, glycerol, is distributed uniformly onan inert support, which is substantially flat, in order to form acrosslinked film.

[0074] The support is inert in that it does not react with theabovementioned compounds and is not involved in the crosslinkingprocess. A hydrophobic support of the PVC or polystyrene type can beused.

[0075] In this case, a solution in which the concentrations of collagen,of hydrophilic additive and of glycerol, if it is present, arepreferably between 2 and 6% in the case of collagen, 0.6 and 2% in thecase of the hydrophilic additive, and 0.3 and 0.8% in the case of theglycerol, is applied to the support.

[0076] The thin layer which is applied advantageously has a density offrom 0.05 to 0.3 g/cm².

[0077] Once the reaction is complete, the film is separated from thesupport.

[0078] The collagenous material according to the invention containscollagen and at least one hydrophilic macromolecular additive inaccordance with a collagen/hydrophilic additive(s) ratio of from 1/1 to9/1, preferably of from 2/1 to 4/1 and, still more preferably, of 3/1.

[0079] The collagenous material is stable at ambient temperature andremains stable for a time which is adequate for manipulating it, inaqueous medium, at temperatures of up to 37 to 40° C.

[0080] The collagenous material according to the invention can be usedfor preventing post-operative adhesions.

[0081] It can be prepared in a “ready-to-use” form by, for example,cutting a film such as described above to the dimensions which areappropriate for the envisaged application and packing it under sterileconditions.

[0082] When the material is prepared in the form of a film, it isparticularly suitable for this application, in particular forsimplifying and speeding up the surgical procedure.

[0083] This is because the collagenous film which is obtained can bemanipulated with ease because it is not sticky to the touch in the drystate and it does not stick to instruments.

[0084] Furthermore, it has an increased density and mechanicalresistance while at the same time being relatively pliable, as isrequired.

[0085] The glycerol improves the pliability of the final materialobtained and can thus facilitate its use.

[0086] The collagenous material according to the invention is able todevelop adhesive properties in aqueous medium.

[0087] After having been implanted in a patient, it can adhereadequately to the organ to be protected, thereby enabling it to remainin place, and is for this reason suitable for being used as apost-operative anti-adhesion barrier. Thus, on contact with the tissues,an inflow of tissue water results in immediate adherence of thecollagenous film. The inflow of water progressively swells the film ofcollagen, whose crosslinking is of sufficiently low density to permit acertain degree of mobility and flexibility of the chains which make upthe film.

[0088] A gel of collagen, which is oxidized, crosslinked and veryhydrated due to the hydrophilic additive which is trapped in the meshesof the gel, is thus formed within a few minutes to a few hours.

[0089] This gel acquires adhesive properties which are adequate forestablishing satisfactory adherence of the biomaterial to the wound tobe protected.

[0090] Furthermore, the hydrophilic macromolecular additive disappears,by diffusion through the oxidized and crosslinked collagenous material,in a few days, with the swelling of this material favouring itsdegradation within less than a month, if not to say less than 7 days, inparticular in 2 to 3 days.

[0091] If the degree of crosslinking of the collagenous material isincreased, in particular by means of incubation at 37° C. in a moistenvironment, before sterilizing by irradiation, it is possible toincrease the resorption time to up to 24 weeks.

[0092] This resorption time of between 1 and 4 weeks is attractive forsome applications in which cicatrization of the damaged tissues takesplace more slowly.

[0093] The invention will be described in more detail with the aid ofthe examples, which are given below by way of illustration and which arenot limiting.

EXAMPLES Example 1

[0094] Preparation of an Acid Precipitate of Collagen which has beenModified by Oxidative Cleavage Using Periodic Acid and Which is notCrosslinked.

[0095] This example is carried out directly on the basis of thepreviously cited patent of TARDY et al. (1994).

[0096] The collagen employed is bovine type I collagen, which isextracted from calf derm by solubilization at acid pH or by digestionwith pepsin and purified by salt precipitations using the techniqueswhich have already been described.

[0097] The products marketed by COLLAGEN Corp. under the names VITROGEN®or ZYDERM® may be used for this application.

[0098] Preference is given to using dry collagen fibres which areobtained by precipitating an acid solution of collagen by means ofadding NaCl and then washing and drying the precipitate which isobtained with aqueous solutions of acetone whose concentration increasesfrom 80% to 100%.

[0099] Human type I or type III collagens, or a mixture of thesecollagens in any proportions, can be used in the same way.

[0100] A solution of 30 g of collagen/l is prepared by dissolving thecollagen in 0.01 N HCl. The volume of the solution is 49 liters.Periodic acid is added to it up to a final concentration of 8 mM, thatis 1.83 g/l.

[0101] The oxidation is carried out at ambient temperature in thevicinity of 22° C. for 3 hours in the absence of light.

[0102] An equal volume of a solution of sodium chloride is then added tothe collagen solution in order to obtain a final concentration of 41 gof NaCl/l.

[0103] After waiting for 30 minutes, the precipitate is collected bydecanting through a wire mesh screen of a porosity of about 100 micronsand then washed 4 times with a solution of 41 g of NaCl/l in 0.01 N HCl.19 kg of acid saline precipitate are obtained. These washes eliminateall traces of periodic acid or iodine-containing derivatives which areformed during the oxidation of the collagen.

[0104] Several washes in an 80% aqueous solution of acetone thenconcentrate the collagen precipitate and remove the salts which arepresent.

[0105] A final wash in 100% acetone results in the preparation of 3.6 kgof a very dense acetone precipitate of acid oxidized collagen which isnot crosslinked and which does not contain any other trace of anundesirable chemical product.

Example 2

[0106] Preparation of Anti-post-operative Adhesion Collagenous FilmsAccording to the Invention.

[0107] The acetone paste prepared as described in Example 1 is taken upin apyrogenic distilled water at 40° C. in order to obtain a collagenconcentration of 3%.

[0108] The solution, of 44 liters in volume, is heated at 50° C. for 30minutes and then filtered under sterile conditions through a membranehaving a porosity of 0.45 microns into a tank at 40° C.

[0109] A sterile, concentrated solution of PEG 6000 (polyethylene glycolhaving a molecular weight of 6000 daltons) is added to this solution at30° C. in order to achieve a PEG concentration of 1%, followed byglycerol in order to achieve a glycerol concentration of 0.6%. The pH ofthe solution is adjusted to 7.0 by adding concentrated sodium hydroxidesolution.

[0110] The volume of the solution is then adjusted with sterile water toobtain final concentrations of collagen, PEG and glycerol of 2.7%, 0.9%and 0.54%, respectively, and then distributed in a thin layer, having adensity of 0.133 g/cm², on a flat hydrophobic support of the PVC orpolystyrene type. The surfaces are then exposed to a sterile stream ofair at ambient temperature, leading to evaporation in approximately 18hours.

[0111] The film which is obtained detaches very readily from thesupport. It can easily be cut to the dimensions which are required forthe experiment.

[0112] After that, in order to conform with the pharmaceuticalregulations and to improve stability during storage, the film isinserted into a double airtight bag.

[0113] The whole is sterilized by beta irradiation and receives a dosewhich is greater than or equal to 25 kilogreys.

[0114] In general, the dried film still contains residual water up to aconcentration which can be as much as 20%.

[0115] A typical composition in the example under consideration is 60%by weight of collagen, 20% by weight of PEG, 12% by weight of gylceroland 8% by weight of water.

[0116] The film which is obtained is stable at ambient temperature. Itremains stable and easily manipulable after being incubated for 2 hoursin water at 37° C.

[0117] It is not essential for glycerol to be present in the material;the glycerol improves the flexibility of the film and makes the filmeasier to use.

[0118] This example can be carried out by replacing the PEG 6000 withPEG 3000 or PEG 4000, with soluble starch (OS France catalogue referenceNo. A2620) or with Dextran T40 (PHARMACIA Fine Chemicals catalogue,Uppsala, Sweden) or with carboxymethyl cellulose. The concentrationsemployed, and the mode of operation, are identical.

[0119] Study of the Properties of the Collagenous Film According to theInvention in Preventing Post-operative Adhesions.

[0120] Toxicology

[0121] The collagen/PEG film produced as described in Example 2 (or aground-up preparation of this film) gives satisfactory results instandardized toxi-cological tests to which biomaterials for implantationare subjected:

[0122] absence of mutagenic character in the AMES test

[0123] normal, moderate reaction in the delayed hypersensitivity testcarried out on guinea-pigs (class III reaction)

[0124] absence of systemic toxicity in mice and rats when administeredby the intravenous or intraperitoneal route

[0125] degradation:

[0126] in less than 7 days when administered to the rat by thesubcutaneous route

[0127] in 2 to 3 days when administered to the rat by theintraperitoneal route.

[0128] Anti-post-operative Adhesion Properties

[0129] The anti-post-operative adhesion properties were analysed usingthe protocol described in the HARRIS et al. (1995) reference.

[0130] The protocol described in this publication was carried out ongroups of 10 rats.

[0131] The tests consist in abrading and dehydrating 2 cm² areas of theperitoneal wall and caecum which are in contact with each other.

[0132] The control group of rats is not given any product for protectingthe wounds which have been created in this way. This group is comparedwith the group of rats which is given a collagen/PEG film as describedin Example 2, which film in each case completely covers the wound andextends 5 mm beyond it over the external non-abraded areas.

[0133] After a wait of 7 days, in conformity with the publishedprotocol, the results are clear-cut:

[0134] no adhesion between the two damaged surfaces is observed in therat group which is treated with the film of the invention, and thecicatrization of each initial wound is complete

[0135] no trace of the film is left.

[0136] Each of the 10 rats in the control group, which was not treatedwith the film of the invention, exhibits adhesions, with thecharacteristics of these adhesions being identical to the resultspublished in the abovementioned HARRIS et al. (1995) document.

[0137] When the biodegradable films which were produced as described inExample 2 were tested on other animal models in other operativeprotocols (WISEMAN 1992 and 1994), they were demonstrated to beeffective in preventing adhesions in applications relating toneurosurgery (in particular herniated discs and vertebrallaminectomies), to cardiac surgery and to gynaecology, in particularuterine surgery. The invention therefore also relates to using thematerials according to the invention in these surgical indications aswell as in orthopaedic surgery (in particular in relation to thetendons) and in ophthalmic surgery.

Example 3

[0138] Preparation of a Collagenous Film in Accordance with AnotherEmbodiment of the Invention

[0139] A film is prepared as described in Example 2.

[0140] After having been separated from its support and cut, each filmis individually packed in a primary bag which is permeable to vapour andthen incubated for 12 hours in an incubator which is at 37° C. and whosedegree of relative humidity is greater than 80%.

[0141] The previous bag is then inserted into a secondary, airtightpackaging which is then sterilized as in Example 2.

[0142] The material is degraded within 2 to 4 weeks by being implantedsubcutaneously or intraperitoneally in rats.

[0143] The film according to this example is degraded less rapidly inthe organism than is the film of Example 2. It is observed that the filmis less adherent to the tissue wound and that it swells less inphysiological medium. Because of this, a material of this nature wouldpreferably be indicated in surgical applications where the risk of thematerial being displaced is low. For other applications, it would bepreferable to attach the film using appropriate means (glue, sutures,etc.). Under these conditions, the same anti-adhesion properties areobserved in vivo.

REFERENCES

[0144] 1. DI ZEREGA G. S. (1994)—“Contemporary adhesion prevention”Fertility and Sterility. 61(2), 219-235.

[0145] 2. HARRIS E. S.—MORGAN R. F.—RODEHEAVER G. T. (1995)—“Analysis ofthe kinetics of peritoneal adhesion formation in the rat and evaluationof potential antiadhesive agents”, Surgery, 117(6), 663-669.

[0146] 3. BECKER J. M.—DAYTON M. T.—FAZIO V. W.—BECK D. E.—STRYKER S.J.—WEXNER S. D.—WOLFF B. G.—ROBERTS P. L.—SMITH L. E.—SWEENEY S.A.—MOORE M. (1996)—“Prevention of postoperative abdominal adhesions by asodium hyaluronate-based bioresorbable membrane: A prospecive,randomized, double-blind multicenter study”, The J. of Amer. College ofSurgeons, 183(4), 297-306.

[0147] 4. KHOURY W.—ABDUL-MALAK N.—HUC A. (1994)—“Membrane collagéniqueanti-adhérence post-opératoire (Collagenous antipost-operative adhesionmembrane)”, patent application No. 94 06995 of Jun. 8, 1994 in France(published under No. 2 720 945).

[0148] 5. TAYOT J. L.—MARESCAUX J.—DUMAS H.—TARDY M. (1988)—“Visceralsurgery patch”, U.S. Pat. No. 5,201,745, issued on Apr. 13, 1993 (underthe priority of French patent application No. 88 03321 of Mar. 15, 1988,issued on Jul. 13, 1990 under No. 2 628 634).

[0149] 6. ORLY I. (1994)—“Use of collagen membranes as peritonealrenewing prostheses”, international patent application WO 96/082 77(under the priority of French patent application No. 94 11 015 of Sep.15, 1994).

[0150] 7. YEUNG J. E.—CHU G. H.—DE LUSTRO F. A.—RHEE W. M. (1995)—“Antiadhesion films and compositions for medical use”, European patentapplication No. 96 102 339.7 (published under No. 0 732 110) (under thepriority of American patent application Ser. No. 403,360 of Mar. 14,1995).

[0151] 8. TARDY M.—TIOLLIER J.—TAYOT J. L. (1994)—“Composition adhésive,à usage chirurgical, à base de collagène modifié par coupure oxydativeet non réticulé (Adhesive composition for surgical use, based oncollagen which is modified by oxidative cleavage and which is notcrosslinked)”—French patent application No. 94 00715 of Jan. 24, 1994,issued on Aug. 2, 1996 under No. 2 715 309.

[0152] 9. TARDY M.—TAYOT J. L. (1986)—“Process for the treatment ofcollagen, notably for facilitating its cross-linking, and the collagenobtained by the application of the said process”, U.S. Pat. No.4,931,546 issued on May 6, 1990 (under the priority of French patentapplication No. 86 10 160 of Jul. 7, 1986, issued on Dec. 5, 1989 underNo. 2 601 371).

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1. Biocompatible collagenous material which is non-toxic andbiodegradable in less than one month, characterized in that it comprisescollagen and at least one hydrophilic macromolecular additive which ischemically non-reactive towards the collagen, with the collagen havingat least partially lost its helical structure and being crosslinked. 2.Collagenous material according to claim 1 , characterized in that it isbiodegradable within less than one week.
 3. Collagenous materialaccording to either of claims 1 and 2, characterized in that thecollagen was modified by being heated above 37° C.
 4. Collagenousmaterial according to one of claims 1 to 3 , characterized in that itcomprises collagen which has been modified by oxidative cleavage andcrosslinked in the presence of at least one hydrophilic macromolecularadditive which is chemically non-reactive towards the said collagen. 5.Collagenous material according to claim 4 , characterized in that thecollagen is modified by oxidative cleavage using periodic acid or one ofits salts.
 6. Collagenous material according to one of claims 1 to 3 ,characterized in that it is crosslinked by beta irradiation or gammairradiation in the presence of a hydrophilic macromolecular additivewhich is chemically non-reactive towards the collagen.
 7. Collagenousmaterial according to any one of the preceding claims, characterized inthat the hydrophilic macromolecular additive has a molecular weightgreater than 3000 daltons.
 8. Collagenous material according to any oneof the preceding claims, characterized in that the hydrophilic additiveis a hydrophilic polymer having a molecular weight of between 3000 and20,000 daltons.
 9. Collagenous material according to any one of thepreceding claims, characterized in that the hydrophilic additive ispolyethylene glycol.
 10. Collagenous material according to any one ofclaims 1 to 7 , characterized in that the hydrophilic additive is apolysaccharide which is preferably selected from starch, dextran andcellulose.
 11. Collagenous material according to any one of thepreceding claims, characterized in that it comprises collagen and atleast one hydrophilic macromolecular additive in accordance with acollagen/hydrophilic additive(s) ratio of from 1/1 to 9/1, preferably offrom 2/1 to 4/1 and, still more preferably, of 3/1.
 12. Collagenousmaterial according to any one of the preceding claims, characterized inthat. it additionally contains glycerol.
 13. Collagenous materialaccording to any one of the preceding claims, characterized in that itis present in the form of a film.
 14. Collagenous material according toany one of claims 1 to 13 , characterized in that it is potentiallyadhesive.
 15. Collagenous material according to one of claims 1 to 5 and7 to 14, characterized in that the material is subjected to irradiationwith beta radiation.
 16. Collagenous material according to claim 15 ,characterized in that the material is subjected to beta irradiation at adose which is greater than or equal to 25 kilogreys.
 17. Process forobtaining a biocompatible collagenous material which is non-toxic,potentially adherent and biodegradable in less than one month.preferably in less than one week. characterized in that it comprises: a)preparing a collagen solution; b) treating the said solution in order tocause the collagen at least partially to lose its helical structure; c)mixing the resulting collagenous solution with a solution containing atleast one hydrophilic macromolecular additive which is chemicallynon-reactive towards the collagen which is present; d) crosslinking thesaid mixture in order to obtain the desired collagenous material. 18.Process according to claim 17 , characterized in that a solution ofcollagen which is modified by oxidative cleavage is prepared in step a).19. Process according to claim 18 , characterized in that, in step a),an acid solution of collagen having a concentration of between 5 and 50g/l is prepared and then mixed, at ambient temperature, with a solutionof periodic acid or one of its salts at a concentration of between 1 and10⁻⁵ M.
 20. Process according to any one of claims 17 to 19 ,characterized in that, in step b), the solution of collagen is treatedby being heated at a temperature greater than 37° C.
 21. Processaccording to claim 20 , characterized in that, in step b), the solutionof collagen is heated at a temperature of between 40 and 50° C. 22.Process according to any one of claims 17 to 21 , characterized in that,in step c), the hydrophilic additive has a molecular weight which isgreater than 3000 daltons.
 23. Process according to any one of claims 17to 22 , characterized in that, in step c), the hydrophilic additive is ahydrophilic polymer having a molecular weight of between 3000 and 20,000daltons.
 24. Process according to any one of claims 17 to 23 ,characterized in that, in step c), the hydrophilic additive ispolyethylene glycol.
 25. Process according to any one of claims 17 to 24, characterized in that, in step c), the hydrophilic additive is apolysaccharide which is preferably selected from starch, dextran andcellulose.
 26. Process according to any one of claims 17 to 25 ,characterized in that, in step c), the concentration of hydrophilicadditive is from 2 to 10 times lower than that of the collagen. 27.Process according to any one of claims 17 to 26 , characterized in that,in step c), glycerol is additionally added.
 28. Process according toclaim 27 , characterized in that the concentration of glycerol isbetween 3 and 8 g/l.
 29. Process according to any one of claims 18 to 28, characterized in that, in step d), the mixture is neutralized toneutral pH.
 30. Process according to anyone of claims 17 and 20 to 28,characterized in that, in step d), the mixture is crosslinked by beta orgamma radiation.
 31. Process according to anyone of claims 17 to 29 ,characterized in that the collagenous material derived from step d) issubjected to irradiation with beta radiation.
 32. Process according toclaim 31 , characterized in that the collagenous material is subjectedto beta irradiation at a dose which is greater than or equal to 25kilogreys.
 33. Process according to any one of claims 17 to 32 ,characterized in that a film is formed from the solution containingcollagen, which has at least partially lost its helical structure andwhich may have been modified by oxidative cleavage, at least onehydrophilic additive and, where appropriate, glycerol.
 34. Processaccording to claim 33 , characterized in that a thin layer of thesolution is substantially uniformly applied to an inert support which issubstantially flat, the collagen is allowed to crosslink and the filmwhich has formed is then separated from the support.
 35. Processaccording to either of claims 33 and 34, characterized in that thesolution contains from 2 to 6% collagen, from 0.6 to 2% hydrophilicadditive and from 0.3 to 0.8% glycerol, and the thin layer which isapplied to the said inert support has a density of between 0.05 and 0.3g/cm².
 36. Process according to any one of claims 12 to 24 ,characterized in that the crosslinked collagen is subjected to anincubation at 37° C. in a moist environment before the step of beingsterilized by irradiation, with the material obtained beingbiodegradable within 2 to 4 weeks.